Genome integrity is threatened by DNA double strand breaks (DSBs), which, if left unrepaired, can lead to permanent cell cycle arrest or death. Consequently, complex mechanisms exist for the efficient detection and repair of DNA ends created by DSBs. DNA ends are also encountered at natural chromosome termini, which, conversely, must be protected from DSB repair activities, such as nonhomologous end joining (NHEJ), in order to preserve genome integrity. This is achieved through the specialized nucleoprotein structures known as telomeres. It is now clear that many of the activities that function in response to DNA DSBs also function in normal telomere structure, function, and maintenance. One such protein is the Ku heterodimer, a high affinity DNA end binding complex crucial for NHEJ and, notably, multiple aspects of telomere biology, such as the protection of telomeres from aberrant repair activities, the regulation of telomere length, and the formation of a repressive telomeric chromatin structure, which results in the transcriptional silencing of nearby genes, known as telomeric silencing. Paradoxically, Ku is also a principal effector of the catastrophic end-to-end fusions that can occur at dysfunctional telomeres. How Ku's NHEJ activity is inhibited at wild type telomeres remains poorly defined. Previous work by the PI and others has firmly established that Ku performs distinct activities at DSBs vs. telomeres, however the mechanisms of action at these sites have yet to be fully elucidated. Recently, the PI and co-workers have developed a `two-face'model for Ku's functions at DSBs and telomeres, in which there is an outward face, oriented toward the DNA terminus, which mediates NHEJ, and an inward face, oriented toward telomeric chromatin when bound to a telomere, which mediates telomeric functions. The overall goal of the proposed work is to elucidate the molecular determinants of Ku's activities at telomeres in the model organism, Saccharomyces cerevisiae, thereby expanding and testing the two-face model. Specific Aim 1 will a) further define Ku's inward face, particularly with respect to Ku's telomere end protection property, via site-directed mutagenesis;b) determine whether one or more of Ku's telomeric activities require DNA end binding by generating and characterizing DNA end binding defective Ku proteins;and 3) determine the role of end binding in protecting broken as compared to telomeric ends by analyzing the properties of Ku mutants consisting of solely the DNA binding core. Specific Aim 2 will identify and characterize proteins that interact with Ku in telomere end protection or other telomeric functions using genetic and biochemical approaches. Specific Aim 3 will further define the function at Ku's repair-specific outward face by identifying the factor(s) that interact with an NHEJ-specific surface 1-helix it contains;these will include NHEJ-factors as well as telomeric factors that may inhibit Ku-mediated NHEJ at telomeres. Thus, through a combination of genetic and molecular approaches, this proposal offers to make a substantial contribution to the field's current understanding of the function of Ku, which may inform studies in human cells, where Ku is essential.